AMINOPEPTIDASE-A FROM RHYNCHOSCIARA-AMERICANA (DIPTERA) LARVAL MIDGUTS - PROPERTIES AND MIDGUT DISTRIBUTION

L-aspartic acid alpha-(beta-naphthylamide) (Asp beta NA) hydrolase act ivity is restricted mostly to the midgut caeca of Rhynchosciara americ ana larvae. The membrane-bound activity is solubilized in detergent an d, after electrophoretic separation, proved to be identical to leucine p-nitroanilide (LpNA) hydrolases previously described. Differential c entrifugation of midgut caeca homogenates, followed by assays of enzym e markers and aminopeptidase, suggests that the soluble Asp beta NA hy drolase is associated with the cell glycocalyx. Soluble aminopeptidase s from R. americana midgut caeca are resolved into three fractions by gel electrophoresis. The slow migrating fraction hydrolyzes Asp beta N A well and displays a low activity on LpNA and proline beta-naphthylam ide (Pro beta NA). Thus, this enzyme is an ami; nopeptidase A (EC 3.4. 11.7). It has a pH optimum of 7.5, M(r) 117,000 (gel filtration), and is competitively inhibited by aspartate hydroxamate (K-i 0.1 mM). Neve rtheless, this enzyme, in contrast to the vertebrate enzyme, is not ac tivated by calcium ions. The aminopeptidase A seems to have a charge v ariant that displays an intermediate migration and is not resolved fro m an aminopeptidase N (enzyme very active on LpNA). These two activiti es are not resolved by either gel filtration or ion-exchange chromatog raphy. The aminopeptidases N with intermediate and high migration, pre viously reported to be charge variants, were shown in this paper to di ffer in substrate specificities and in the strength with which they as sociate to the cell glycocalyx. (C) 1994 Wiley-Liss, Inc.